Roles of Chaperone/Usher Pathways of Yersinia pestis in a Murine
Model of Plague and Adhesion to Host Cells
Matthew Hatkoff,a,bLisa M. Runco,cCeline Pujol,a,b* Indralatha Jayatilaka,a,dMartha B. Furie,a,dJames B. Bliska,a,band
David G. Thanassia,b
Center for Infectious Diseases,aDepartment of Molecular Genetics and Microbiology,band Department of Pathology,dStony Brook University, Stony Brook, New York,
USA, and Department of Life Sciences, New York Institute of Technology, Old Westbury, New York, USAc
Yersinia pestis and many other Gram-negative pathogenic bacteria use the chaperone/usher (CU) pathway to assemble viru-
lence-associated surface fibers termed pili or fimbriae. Y. pestis has two well-characterized CU pathways: the caf genes coding for
the F1 capsule and the psa genes coding for the pH 6 antigen. The Y. pestis genome contains additional CU pathways that are
structed deletion mutations in the usher genes for six of the additional Y. pestis CU pathways. The wild-type (WT) and usher
pestisstrainscontainingdeletionsinCUpathways y0348-0352,y1858-1862,andy1869-1873 wereattenuatedforvirulencecom-
during pneumonic plague. We examined binding of the Y. pestis WT and usher deletion strains to A549 human lung epithelial
cells, HEp-2 human cervical epithelial cells, and primary human and murine macrophages. Y. pestis CU pathways y0348-0352
andy1858-1862 werefoundtocontributetoadhesiontoallhostcellstested,whereaspathway y1869-1873 wasspecificforbind-
mutants identifies three of the additional CU pathways of Y. pestis as mediating interactions with host cells that are important
is maintained in a number of natural reservoirs, including rats,
are primarily spread through the bite of infected arthropods, in-
cluding, most notably, the rat flea, Xenopsylla cheopis (37, 38, 74).
Y. pestis introduced into a human host through the bite of an
infected flea can enter the bloodstream and spread to a regional
and initiate an inflammatory response. This inflammation causes
the lymph node to swell and transform into a painful black bubo,
the hallmark of bubonic plague (53). Y. pestis can re-enter the
bloodstream from the lymph node, leading to septicemic plague
by this route results in a secondary pneumonic plague, which can
then lead to direct human-to-human spread of the bacteria via
respiratory droplets. Direct inhalation of Y. pestis results in a pri-
mary pneumonic plague. Humans infected with all forms of
vention (14, 53).
Y. pestis evolved from the enteric pathogen Y. pseudotubercu-
losis 1,500 to 20,000 years ago (2). Key to its adaptation to the
vector-borne lifestyle was the acquisition of two Y. pestis-specific
plasmids, pMT1 and pPCP1 (9). The pPCP1 plasmid encodes the
Pla plasminogen activating protease, which is important for the
ability of Y. pestis to proliferate and persist within the lungs in
pneumonic form and for its dissemination from the site of infec-
tion in bubonic form (9, 45, 62). Pla also contributes to the bac-
teria’s ability to adhere to and invade host cells, making it one of
the adhesins expressed by Y. pestis (4, 18). The second pestis-spe-
cific plasmid, pMT1, contributes to both transmission through
ersinia pestis is a facultative intracellular, Gram-negative bac-
terial pathogen that causes the deadly disease plague. Y. pestis
fleas and increased virulence in the human host. The caf genes
present on pMT1 code for expression of the fraction 1 (F1) cap-
sule. The F1 capsule, which is expressed at 37°C, is a major pro-
tective antigen of Y. pestis and forms a dense coating around the
vector and contributes to pathogenesis in the mammalian host
(61, 73). Y. pestis also contains a third plasmid, pCD1, that is
shared with enteropathogenic Yersinia spp. (Y. pseudotuberculosis
and Y. enterocolitica) and is critical for virulence. The pCD1 viru-
lence plasmid encodes the type three secretion system (T3SS) and
specific growth conditions and allows the direct injection of Yops
into the cytoplasm of target host cells, causing a variety of effects,
including modulation of the host immune response, blocking
phagocytosis, and triggering host cell death (5, 7, 15, 16, 32, 65).
Adhesins are critical virulence factors of pathogenic bacteria,
mediating interactions with host cells and allowing colonization
of specific sites within the host (55). For the pathogenic Yersinia,
adhesin-mediated host cell binding is also important to facilitate
Received 26 April 2012 Returned for modification 21 May 2012
Accepted 18 July 2012
Published ahead of print 30 July 2012
Editor: A. J. Bäumler
Address correspondence to David G. Thanassi, email@example.com.
*Present address: Celine Pujol, DGA Maîtrise NRBC, Le Bouchet, Vert le Petit,
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
iai.asm.org Infection and Immunityp. 3490–3500 October 2012 Volume 80 Number 10
the delivery of Yop effector proteins via the pCD1-encoded T3SS.
the enteropathogenic Yersinia: YadA and Inv (18, 41, 59, 63). Al-
ternate adhesins have been identified in Y. pestis, including the
outer membrane protein Ail and the autotransporter proteins
YapC and YapE (26, 27, 46). In addition, the Y. pestis genome
contains 10 gene clusters belonging to the chaperone/usher (CU)
system present in Gram-negative bacteria that is dedicated to the
in adhesion, termed pili or fimbriae (66, 71, 77). The functions of
two of the Y. pestis CU pathways have been extensively character-
ized: the plasmid pMT1-encoded caf system that expresses the F1
capsule as described above and the psa genes coding for the pH 6
antigen. The pH 6 antigen is typically expressed at 37°C under
low-pH conditions and forms thin, flexible fibers on the bacterial
surface (48, 49). The pH 6 antigen functions in adhesion to host
cells and has been shown to bind phosphatidylcholine on lung
epithelial cells but also plays a role in evasion of phagocytosis (25,
33, 40, 49). The pH 6 antigen is a virulence factor of Y. pestis and
contributes to the pathogenesis of both bubonic and pneumonic
plague (47, 73).
Biogenesis of pili by the CU pathway relies on a periplasmic
chaperone and an integral outer membrane protein termed the
(54, 66, 71, 77). The usher genes for two of the Y. pestis CU path-
ways (y1539-1544 and y4060-4063) are disrupted by an insertion
sequence or premature stop codon (Fig. 1), and thus these path-
ways are not expected to be functional. The caf and psa genes
belong to the FGL subfamily of CU pathways that assemble thin,
flexible fibers (77). In contrast, the additional CU pathways of Y.
pestis belong to the FGS subfamily that assembles rigid, rod-like
adhesive pili. In agreement with this, Y. pestis was shown to ex-
press pilus fibers distinct from the F1 capsule and pH 6 antigen
(60). Moreover, a recent study by Felek et al. demonstrated that
heterologous expression of the six intact additional Y. pestis CU
pathways in Escherichia coli resulted in the assembly pilus-like
0352, y0561-0563, y1858-1862, and y3478-3480 enhanced biofilm
formation by E. coli (25). However, when deletion mutations of
the CU pathways were constructed in the Y. pestis KIM5 strain,
only loss of the psa locus, coding for the pH 6 antigen, resulted in
decreased adhesion to host cells and decreased biofilm formation
(25). Finally, Felek et al. found that a Y. pestis KIM5 strain con-
taining a deletion of CU pathway y1858-1862 was attenuated for
to the parental wild-type (WT) strain (25). Taken together, these
ute to virulence. However, the functions of the Y. pestis CU path-
ways in host-pathogen interactions and in the pathogenesis of
plague remain to be established.
In the present study, we tested the roles of the CU pathways of
Y. pestis in virulence, using the murine bubonic (subcutaneous)
the usher gene for each of the six intact additional CU pathways
ground. Comparison of these mutants with WT KIM5? revealed
that CU loci y0348-0352, y1858-1862, and y1869-1873 contribute
to virulence via the intranasal, but not subcutaneous, routes of
infection. We found no differences between WT Y. pestis and
usher deletion mutants for biofilm formation or autoaggregation
in vitro. However, Y. pestis KIM6? usher mutants containing de-
The caf (F1) and psa (pH 6) CU gene clusters are shown at the bottom of the figure. Usher gene y1543 is disrupted by an insertion sequence (IS). The usher for
pathway y4060-4063 is disrupted by a frameshift mutation into two open reading frames (y4061 and y4062).
Chaperone/Usher Pathways of Yersinia pestis
October 2012 Volume 80 Number 10 iai.asm.org 3491
letions in CU loci y0348-0352, y1858-1862, and y1869-1873 were
defective for adhesion to host cells compared to the WT strain.
The Y. pestis CU pathways found to function in host cell binding
sal infection, establishing a function for at least three of the addi-
tional CU pathways in Y. pestis-host interactions and the patho-
genesis of plague.
MATERIALS AND METHODS
KIM, a biovar 2.MED strain (1). Y. pestis KIM6? is an attenuated pgm-
is fully virulent and contains both the pgm locus and the pCD1 virulence
plasmid marked with an ampicillin resistance (Ampr) gene (34). Specific
growth conditions for Y. pestis are noted for each experiment. E. coli
strains were grown in LB medium at 37°C with aeration. When needed,
100 ?g/ml, kanamycin (Kan) at 50 ?g/ml, or chloramphenicol (Cml) at
topyranoside) was added to 50 ?M final concentration to induce expres-
sion of plasmid-encoded genes when necessary.
The usher genes were deleted in Y. pestis KIM6? using the pKOBEG-
sacB system (20, 22). Briefly, the pKOBEG-sacB plasmid was first intro-
duced into KIM6? by electroporation. A DNA fragment containing a
amplified by PCR with the primer pairs y(usher gene)_kanF and y(usher
gene)_kanR (Table 2). Approximately 1 to 2 ?g of purified PCR product
was used to electroporate KIM6?/pKOBEG-sacB cells. Kanrtransfor-
mants harboring the mutated allele were isolated and screened by PCR.
Plasmid pKOBEG was cured by subculturing the bacteria at 26°C on me-
(usher gene)::kan strain was then transformed with plasmid pFLP2 by
conjugation with E. coli strain S17?pir, and the Kanrcassette was excised
using the flanking FRT sites. To cure pFLP2, bacteria were subcultured at
clones. These final strains were designated KIM6? ?(usher gene) (Table
1). All usher gene deletions were confirmed by PCR using appropriate
For construction of complementing plasmids py0350, py1858, and
py1871 (Table 1), the usher genes were amplified from KIM6? by PCR
using Taq polymerase (Invitrogen) and the primer pairs listed in Table 2.
The PCR products were ligated into plasmid pGEM-T Easy (Promega),
the resulting plasmids were then digested with EcoRI and BamHI or
BamHI and SalI, and the fragments encoding the usher genes were puri-
ments were then ligated into plasmid pMMB91 that had been similarly
digested and purified. Ligation products were transformed into E. coli
genes downstream of the IPTG-inducible Ptacpromoter, were confirmed
by sequencing. Purified plasmids were then transformed into their cog-
nate KIM6? usher deletion strains.
To generate the usher deletion mutations in the fully virulent Y.
pestis KIM5? background, plasmid pCD1Ap was introduced by elec-
TABLE 1 Strains and plasmids used in this study
Strain or plasmid Relevant characteristic(s)a
Source or reference
Cloning strain; hsdR recA endA
Strain for maintenance of conditional replicative oriR? plasmids
Strain used for conjugation
Attenuated biovar 2.MED strain; pgm?, pCD1?
Deletion of usher gene y0350 in KIM6?
Deletion of usher gene y0562 in KIM6?
Deletion of usher gene y1858 in KIM6?
Deletion of usher gene y1871 in KIM6?
Deletion of usher gene y2390 in KIM6?
Deletion of usher gene y3480 in KIM6?
Fully virulent biovar 2.MED strain; KIM6?/pCD1Ap
Deletion of usher gene y0350 in KIM5?
Deletion of usher gene y0562 in KIM5?
Deletion of usher gene y1858 in KIM5?
Deletion of usher gene y1871 in KIM5?
Deletion of usher gene y2390 in KIM5?
Deletion of usher gene y3480 in KIM5?
Cloning vector; Ampr
Expression vector, IPTG-inducible promoter; Kanr
Usher gene y0350 in pMMB91
Usher gene y1858 in pMMB91
Usher gene y1871 in pMMB91
FRT sites; KanrAmpr
? phage red???, arabinose inducible; Clmr
sacB FLP-?pR; Ampr
pCD1 virulence plasmid with bla; Amprcassette
gfp3.1 in vector pMMB207, IPTG inducible; AmprClmr
aClmr, chloramphenicol resistance; Ampr, ampicillin resistance; Kanr, kanamycin resistance.
Hatkoff et al.
iai.asm.orgInfection and Immunity
troporation into the KIM6? usher deletion strains under biosafety
level 3 (BSL3) conditions. The resultant KIM5?Ap strains (Table 1)
were selected by plating onto Yersinia Selective Media (YSM) agar
containing 30 ?g/ml Amp.
Transmission electron microscopy. Y. pestis was grown at 28°C or
resuspended into phosphate-buffered saline (PBS), and then adsorbed to
were fixed with 1% glutaraldehyde for 1 min, washed twice with PBS,
tungstic acid (Ted Pella) for 35 s. The grids were examined on a TECNAI
12 BioTwin G02 microscope (FEI) at an 80-kV accelerating voltage. Dig-
ital images were captured with an AMT XR-60 charge-coupled device
digital camera system (Advanced Microscopy Techniques).
Mouse infection experiments. Mouse infections were performed at
tainment Laboratory (Newark, NJ) under BSL3 conditions. All animal
research protocols were approved by the Institutional Animal Care and
Six- to eight-week-old female C57BL/6 mice (Jackson Laboratories)
were utilized for the infections. Inoculation via the subcutaneous and
intranasal routes were used to mimic bubonic and pneumonic plague,
respectively. Y. pestis strains were grown overnight at 28°C in HIB, resus-
pended in PBS, and diluted in PBS to achieve the desired infectious dose.
TABLE 2 Primers used in this study
Primer function and nameSequence (5=–3=)
Primers used for usher deletions in Y. pestis
Primers used to confirm usher deletions in Y. pestis
Primers used to amplify usher genes for complementation plasmids
Chaperone/Usher Pathways of Yersinia pestis
October 2012 Volume 80 Number 10iai.asm.org 3493
Groups of five mice were injected subcutaneously with 50 ?l containing
200 to 250 CFU or inoculated intranasally with 25 ?l containing 2,000 to
2,500 CFU (approximately 4 to 5 50% lethal doses [LD50] for the respec-
CFU counts. The mice were observed twice daily and monitored for sur-
vival for 21 days.
Biofilm formation assay. Crystal violet staining was used to deter-
mine biofilm formation and cell attachment to polystyrene as described
by O’Toole et al. (51). Briefly, overnight cultures of Y. pestis were resus-
The OD600of the cultures was read in a microplate reader (SpectraMax).
Bacterial cultures were then washed twice with 100 ?l of PBS, and 0.01%
crystal violet was added. The bacteria were incubated with the crystal
violet for 15 min at room temperature. The bacteria were then washed
solubilized with 80% ethanol and 20% acetone. The absorbance of the
normalized to bacterial culture density.
Autoaggregation assay. Y. pestis strains were grown overnight at ei-
ther 28 or 37°C in HIB. The overnight cultures were resuspended to an
every 20 min.
Tissue culture. A549 human lung epithelial cells were grown in Dul-
becco modified Eagle medium (DMEM; Gibco) containing 10% fetal bo-
vine serum (FBS; HyClone). HEp-2 human epithelial cells were grown in
MEM (Gibco) containing 10% FBS. Murine bone marrow-derived mac-
rophages (muBMDM) were obtained as described previously (57, 58).
Briefly, cells from the femurs of female WT C57BL/6 mice were grown in
bone marrow medium (49% DMEM [Gibco], 30% L-cell supernatant,
20% FBS, 1% sodium pyruvate [Gibco]). When muBMDM were seeded
for infection, they were grown in infection medium (79% DMEM
monocyte-derived macrophages (huMDM) were isolated as described
previously (30) from healthy human donors and directly seeded at 1.5 ?
105cells/well in 24-well plates (Corning) on coverslips. The cells were
allowed to differentiate for 5 days in RPMI medium (Gibco) containing
Bacterial adhesion and invasion assays. Mammalian cells were
seeded onto coverslips in a 24-well plate (Corning) at concentrations of
1.5 ? 105cells per well, followed by incubation overnight at 37°C in 5%
or usher deletion mutant Y. pestis KIM6? strains or the same strains
at 37°C with aeration until the OD600was 0.7. Y. pestis strains containing
the pGFP plasmid (for invasion studies) or deletion strains containing
complementation plasmids were induced for expression of the plasmid-
encoded genes by the addition of IPTG at 2 h of growth. For adhesion
experiments, the bacteria were resuspended to a multiplicity of infection
(MOI) of 50 in the appropriate medium for the host cell type used. For
invasion experiments, bacteria were resuspended to an MOI of 50 for the
epithelial cells or an MOI of 10 for the macrophages. For adhesion exper-
iments, the muBMDM and huMDM were pretreated with 5 ?g of cy-
the plate was centrifuged (50 ? g, 4 min, room temperature) to facilitate
bacterial contact. After 2 h at 37°C in 5% CO2(or 20 min for the macro-
30 min in 2.5% paraformaldehyde at room temperature. All subsequent
steps were completed at room temperature. The cells were blocked with
3% bovine serum albumin (BSA) in PBS for 20 min and then incubated
with rabbit anti-Yersinia antiserum SB349 (6) diluted 1:1,000 in 3% BSA
a secondary goat anti-rabbit antibody conjugated to Alexa Fluor 594 (In-
vitrogen) was added at a 1:2,000 dilution in 3% BSA in PBS, followed by
incubation for 30 min. The cells were then washed again three times with
PBS. The coverslips were mounted on glass slides using ProLong Gold
antifade reagent (Invitrogen), and the slides were examined on a Zeiss
Axioplan2 microscope using a ?40 objective lens. Phase-contrast and
epifluorescence images were captured using a Spot camera (Diagnostic
Instruments) and processed using Adobe Photoshop. For each experi-
the host cells were quantified for each field to calculate the number of
bacteria/cell. The values obtained for the 10 fields were then averaged to
obtain the number of bacteria/cell for the experimental replicate.
Bacterial survival during macrophage infection. muBMDM were
seeded on coverslips in a 24-well plate (Corning) at concentrations of
1.5 ? 105cells per well, followed by incubation overnight at 37°C in 5%
or usher deletion mutant Y. pestis KIM6? strains were diluted 1:20 into
fresh HIB and grown at 37°C with aeration until the OD600was 0.7. The
muBMDM were washed three times with PBS, 1 ml of bacteria with an
MOI of 5 was added to each well, and the plate was centrifuged (50 ? g, 4
min, room temperature) to facilitate bacterial contact. After 20 min at
37°C, 5% CO2the cells were washed once with PBS, and fresh infection
three times with 1 ml of PBS. Fresh medium with or without 2 ?g of
were incubated for an additional 23 h.
in PBS for 10 min at 37°C. The lysates were removed, the wells were
washed with 0.5 ml of PBS, and the lysates and wash were pooled in a
1.5-ml microcentrifuge tube. Serial 10-fold dilutions were then plated on
LB agar, followed by incubation at 28°C for 2 days, and the CFU were
Outer membrane isolation and analysis. KIM6? WT or the usher
at 37°C with aeration until an OD600of ?0.7 was reached. Bacteria were
harvested, washed, resuspended in 1 ml of 20 mM Tris-HCl (pH 8) con-
taining Complete protease inhibitor cocktail (Roche), and lysed by soni-
cation for 2 min (15 s on, 15 s off) in an ice-water bath. Whole bacteria
were removed by centrifugation (8,000 ? g, 2 min, 4°C). Sarkosyl (sodi-
um-N-lauroylsarcosinate; Fisher) was added to the supernatant fraction
room temperature to selectively solubilize the cytoplasmic membrane.
The outer membrane was then pelleted by centrifugation (15,000 ? g, 30
min, 4°C) and resuspended in 0.1 ml of 20 mM Tris (pH 8)–0.3 M NaCl.
An equal volume of 2? sodium dodecyl sulfate (SDS) sample buffer was
added, and the sample was incubated for 10 min at 95°C prior to separa-
tion by SDS-polyacrylamide gel electrophoresis (PAGE). The expression
massie blue-stained SDS-PAGE gels or by immunoblotting with anti-F1
antibody (60) at 1:1,000, anti-Pla antibody (72) at 1:500, anti-PsaA anti-
body (47) at 1:1,500, or anti-Ail antibody (76) at 1:500. Immunoblots
were developed with alkaline phosphatase-conjugated secondary anti-
bodies and BCIP (5-bromo-4-chloro-3-indolylphosphate)/NBT (ni-
troblue tetrazolium) substrate (KPL).
Statistical analysis. Mouse survival curves were compared using the
log-rank test with data obtained from three independent experiments
to six independent experiments with three replicates each. Statistical sig-
tiple-comparison post test. Statistical calculations were performed using
Hatkoff et al.
iai.asm.orgInfection and Immunity
Construction and initial phenotypic characterization of Y. pes-
we constructed a set of deletion mutations in the KIM6? back-
ground (pgm-positive, pCD1-negative) using the lambda Red re-
combination system (20, 22). We deleted the usher gene for each
of the six intact additional CU pathways (Fig. 1), creating strains
KIM6? ?y0350, ?y0562, ?y1858, ?y1871, ?y2390, and ?y3480.
The KIM6? usher deletion mutants showed no growth defects
compared to the parental WT strain when grown in HIB at either
28 or 37°C (data not shown). In addition, no differences were
strain and the usher deletion mutants, nor were differences de-
shown). Y. pestis KIM6? was previously shown to express pilus-
like fibers on its cell surface that were distinct from the F1 capsule
and pH 6 antigen (25, 60). To determine whether one of six CU
the KIM6? usher deletion mutants for the presence of pili by
transmission electron microscopy. Each deletion mutant ex-
pressed pilus fibers similar to the WT strain (data not shown),
suggesting either that more than one of the CU pathways encodes
the pilus fibers or that the fibers are encoded by another, as-yet-
Y. pestis forms biofilms in the flea midgut to allow for effective
transmission of the bacteria from the flea vector to the host (19).
Biofilm formation generally depends on the hms system, which is
found in the pgm locus (8, 37, 53). CU pili contribute to biofilm
sion of four of the Y. pestis CU loci (y0348-0352, y0561-0563,
y1858-1862, and y3478-3480) was previously shown to promote
biofilm formation by E. coli (25, 56, 69). However, disruption of
tion by the Y. pestis KIM5 (?pgm) strain (25). To test whether the
CU pathways might contribute to biofilm formation in the pres-
ence of an intact hms system, we examined our panel of KIM6?
(pgm-positive) usher deletion mutants. The mutant bacteria
formed biofilms similar to WT KIM6? when grown in HIB at 28
or 37°C (data not shown), indicating that none of the six CU
pathways is required for biofilm formation under the experimen-
tal conditions tested. In addition to biofilm formation, Y. pestis
bacteria autoaggregate during growth in culture media at 28°C,
and there is evidence that autoaggregation may correlate with the
deletion mutants and parental KIM6? strain grown in HIB at
28°C for autoaggregation. All bacteria behaved similarly under
the CU pathways is required for Y. pestis autoaggregation. As pre-
due to expression of the F1 antigen (28).
Contributions of the additional CU pathways of Y. pestis in
the murine model of plague. To analyze the roles of the Y. pestis
CU pathways in the host during infection, we first converted the
ground by adding in the pCD1 virulence plasmid by electropora-
tion. As for the KIM6? usher deletion mutants, the KIM5? mu-
tants showed no growth defects compared to the WT strain when
grown in HIB at either 28 or 37°C, and no differences were de-
tected in biofilm formation or autoaggregation (data not shown).
Approximately 4 to 5 LD50of the parental KIM5? strain or equal
doses of the six usher deletion mutants were delivered to mice via
subcutaneous injection (200 to 250 CFU) or intranasal inocula-
tion (2,000 to 2,500 CFU) to mimic bubonic or pneumonic
plague, respectively. No statistically significant differences be-
tween the WT strain and any single usher deletion mutant were
observed in the time to death for mice infected by the subcutane-
ous route (Fig. 2). However, there was a trend toward increased
survival times for the usher deletion mutants compared to WT
FIG 2 Subcutaneous infection of C57BL/6 mice with Y. pestis usher deletion mutants. Mice were infected with WT KIM5? or usher deletion mutants via the
subcutaneous route with 4 to 5 LD50(200 to 250 CFU), and the time to death was recorded. Mice were monitored for signs of illness or death for 21 days. Each
graph represents the combined data from three separate experiments with 5 mice each for a total of 15 mice per strain. There were no significant differences for
any of the deletion mutants compared to the WT strain.
Chaperone/Usher Pathways of Yersinia pestis
October 2012 Volume 80 Number 10iai.asm.org 3495
7, which is typical for this route of infection with fully virulent Y.
pestis (11, 50, 67). The mean time to death for all groups was ?3
days postinfection. Thus, the CU pathways tested do not provide
critical functions during the murine model of bubonic plague.
For the intranasal route of infection, mimicking pneumonic
plague, three of the single usher deletion mutants had significant
shifts in their mouse survival curves compared to the parental
significant (P ? 0.0137) attenuation in Y. pestis virulence, with a
higher percentage of mice surviving past day 3 postinfection (Fig.
3). For the ?y1858 mutant, 7% (1 of 15) of the mice survived the
entire 21-day course of the infection. Mice infected with fully vir-
ulent Y. pestis via the intranasal route typically die between days 3
and 5, with the majority of mice dying on or before day 3 (3, 11,
mutant KIM5? ?y1871 resulted in 13% (2 of 15) of the mice
surviving the course of the infection and an increase in the mean
WT KIM5? (Fig. 3). Mice infected with the other usher deletion
days and had survival curves similar to infection with the WT
0352, y1858-1862, and y1869-1873 function within the host and
contribute to the virulence of Y. pestis during pneumonic plague.
Y. pestis CU pathways y0348-0352 and y1858-1862 mediate
adhesion to human epithelial cells. The virulence attenuation of
the additional CU pathways may mediate interactions with host
cells during infection. We therefore used a microscopy-based as-
say to compare WT KIM6? to the usher deletion mutants for
association with different host cells. We first examined the inter-
line, and HEp-2 cells, a human cervical epithelial cell line. Bacte-
rial binding to the host cells was detected using a polyclonal anti-
Y. pestis antibody in the absence of permeabilization, so only sur-
face-bound bacteria were visualized (Fig. 4A). For A549 cells, the
FIG 3 Intranasal infection of C57BL/6 mice with Y. pestis usher deletion mutants. Mice were infected with WT KIM5? or usher deletion mutants via the
intranasal route with 4 to 5 LD50(2,000 to 2,500 CFU), and the time to death was recorded. Mice were monitored for signs of illness or death for 21 days. Each
graph represents the combined data from three separate experiments with 5 mice each for a total of 15 mice per strain. Mice infected with deletion mutants
?y0350, ?y1858, or ?y1871 were significantly attenuated (*, P ? 0.05; ***, P ? 0.001) compared to mice infected with the WT strain.
FIG 4 Binding of Y. pestis usher deletion mutants to A549 cells. A549 cells
were infected with KIM6? WT, usher deletion mutants, or complemented
strains (denoted with a “C”) at an MOI of 50 for 2 h. The cells were then fixed
secondary antibody conjugated to Alexa Fluor 594 (red). For each infection,
phase-contrast and epifluorescence images from 10 random fields were cap-
tured using a ?40 objective, and the average number of bacteria/cell was cal-
strains. (B) Adhesion data for all strains. The results (bacteria/cell) were cal-
culated from three independent experiments with three replicates per experi-
ment. Bars represent means ? the standard errors of the mean (SEM) (*, P ?
0.05; ***, P ? 0.001 [for comparison of each strain with the WT]).
Hatkoff et al.
iai.asm.org Infection and Immunity
50, adhered at a level of approximately two bacteria per host cell.
No differences were detected in adhesion of the KIM6? ?y0562,
?y1871, ?y2390, or ?y3480 usher deletion mutants compared to
the WT strain; however, usher deletion mutants ?y0350 and
?y1858 both exhibited ?2-fold decreased adherence (Fig. 4B).
Binding of the KIM6? ?y0350 and ?y1858 deletion mutants to
A549 cells was restored back to WT levels upon expression of the
deleted usher gene in trans, demonstrating the specificity of the
usher deletion mutations (Fig. 4B).
A similar result was obtained for binding of the KIM6? WT
and usher deletion mutants to HEp-2 cells. The overall level of
bacterial association with the HEp-2 cells was lower than for the
A549 cells, with ?0.5 bacteria binding per HEp-2 cell for the pa-
rental KIM6? strain. Despite this lower overall binding, 2-fold
and ?y1858 deletion mutants, although the binding defect of the
?y1858 mutant did not reach statistical significance (Fig. 5). No
differences in binding were detected for the other KIM6? usher
deletion mutants, and the complemented ?y0350 and ?y1858
strains bound to the HEp-2 cells at levels indistinguishable from
WT (Fig. 5).
of Y. pestis inside the A549 and HEp-2 cells, similar experiments
were performed with Y. pestis strains expressing green fluorescent
protein (GFP). In these assays, intracellular bacteria appeared
green due to the GFP and extracellular bacteria appeared both
uptake for all Y. pestis strains into the A549 and HEp-2 cells was
very low, with no invasion seen for the A549 cells and approxi-
mately one intracellular bacterium detected for every 10 HEp-2
cells (data not shown). This low level of uptake is in keeping with
in uptake into either host cell type were detected for the parental
KIM6? strain and usher deletion mutants. Given the low-to-ab-
sent levels of bacterial uptake, we conclude that the decreased
association of the Y. pestis ?y0350 and ?y1858 deletion mutants
cell surface. These data identify CU pathways y0348-0352 and
suggest that loss of these interactions may underlie the virulence
attenuation of the corresponding usher deletion mutants in the
Y. pestis CU pathways y0348-0352, y1858-1862, and y1869-
1873 mediate adhesion to primary murine and human macro-
primary murine and human macrophages, muBMDM and
huMDM, respectively. Macrophages are professional phagocytes
at an MOI of 50 (data not shown). As with the A549 and HEp-2
for the parental KIM6? strain and usher deletion mutants. We
also found no differences in the intracellular or extracellular sur-
To specifically measure binding of the Y. pestis strains to the mac-
cytochalasin D prior to infection to block phagocytosis. Under
these conditions, WT KIM6? and usher deletion mutants
?y0562, ?y2390, and ?y3480 adhered at levels of approximately
1.5 and 1.0 bacteria per muBMDM and huMDM, respectively
(Fig. 6). The ?y0350 and ?y1858 usher deletion mutants, which
had decreased binding to the epithelial cells, also exhibited signif-
rophages, with levels approximately 2- to 3-fold lower than to the
WT strain (Fig. 6). Interestingly, usher deletion mutant ?y1871
also showed a similar two-to-3-fold reduction in binding to the
muBMDM and huMDM (Fig. 6). Binding of the three usher de-
ing the specificity of the usher deletion mutations (Fig. 6). Taken
together, these results show that CU pathways y0348-0352 and
y1858-1862 confer binding to receptors present on epithelial cells
and macrophages of both human and murine origin and that CU
pathway y1869-1873 mediates binding to a factor present on hu-
man and murine macrophages but absent from human epithelial
cells. Of note is the fact that the KIM5? ?y1871 usher deletion
mutant had the strongest virulence attenuation observed in the
murine pneumonic plague model, suggesting that Y. pestis-mac-
rophage interactions mediated by pathway y1869-1873 may be
especially important during pathogenesis.
secretion system that assembles adhesive pili associated with vir-
ulence (Fig. 1) (21, 52, 60). Two of these gene clusters, the caf and
tively, have been well studied, and both make important contri-
butions to the pathogenesis of plague (24, 47–49, 61, 73). Of the
eight remaining additional Y. pestis CU pathways, two contain
disruptions to their usher genes and thus are not expected to be
functional (pathways y1539-1544 and y4060-4063; Fig. 1). We fo-
cused here on characterizing roles of the remaining six CU path-
ways in Y. pestis virulence and host-pathogen interactions. Anal-
ysis of single usher deletion mutants in the fully virulent KIM5?
FIG 5 Binding of Y. pestis usher deletion mutants to HEp-2 cells. HEp-2 cells
were infected with KIM6? WT, usher deletion mutants, or complemented
strains as described in Fig. 4. The results (bacteria/cell) were calculated from
resent means ? the SEM (*, P ? 0.05 [for comparison of each strain with the
Chaperone/Usher Pathways of Yersinia pestis
October 2012 Volume 80 Number 10iai.asm.org 3497
the intranasal, but not the subcutaneous, route of infection. Cell
culture infection studies of the single usher deletion mutants in
the KIM6? background (lacking the pCD1 virulence plasmid)
host cells. These results show that at least three of the additional
CU pathways are virulence factors of Y. pestis and function in
adhesion to host cells.
In agreement with our findings, Felek et al. previously identi-
fied CU locus y1858-1862 as contributing to the virulence of Y.
that pili assembled by pathway y1858-1862 may be important for
bubonic, routes, arguing against a general systemic role for path-
way y1858-1862. Future studies will need to address the specific
host receptors recognized by the y1858-1862 pili, as well as the
other Y. pestis CU pili, and determine at what points in the infec-
1873 in virulence (25). There are several differences between the
studies that may explain these results, including that the previous
study used the attenuated KIM5 (?pgm) strain of Y. pestis and
only examined the intravenous route of infection. Although we
it is worth noting that this cluster is located in the pgm locus, and
thus ?pgm strains of Y. pestis lack this CU pathway.
The individual usher deletion mutations did not alter biofilm
formation or autoaggregation by Y. pestis, suggesting that the in
anism. The lack of biofilm or autoaggregation phenotypes is con-
mediating autoaggregation (8, 28, 37, 53). Importantly, we ob-
tained significant phenotypes for adhesion of the KIM6? usher
deletion mutants to host cells. Specifically, compared to the WT
strain, deletions of the usher genes for CU pathways y0348-0352
and primary human and murine macrophages. Deletion of the
decreased binding, but this was specific for the macrophages. The
correlation between the CU pathways identified as important for
actions with host cells during pathogenesis. In contrast to our
results, Felek et al. did not detect any loss in binding to host cells
for Y. pestis KIM5 strains containing deletions of the same CU
epithelial cells and macrophages, the prior study did not examine
binding to A549 cells and used macrophage-like cell lines (RAW
plating assay, as opposed to an MOI of 50 and the microscopy-
based method used here. We have found the microscopy-based
assay to be more sensitive and reproducible for detecting differ-
may have allowed us to detect the 2- to 3-fold changes in binding
strain used by Felek et al. contains the pCD1 virulence plasmid,
and it is possible that under tissue culture conditions the engage-
ment of host cells by the T3SS apparatus may have masked bind-
ing defects caused by loss of the CU pathways.
The decreased binding to host cells for the ?y0350, ?y1858,
virulence for these usher deletion strains was also relatively small;
the ?y1871 strain had the strongest phenotype, with a 1-day in-
multiple CU pathways and other adhesins expressed by Y. pestis.
Although it lacks the major YadA and Inv adhesins expressed by
enteropathogenic Yersinia spp. (59, 63), Y. pestis is known to ex-
press a number of other adhesins, including Pla, Ail, YapC, YapE,
results and consistent with redundant functions, deletion of any
one adhesin has not been found to completely abolish binding to
a given host cell type (18, 25–27, 29, 31, 46, 49). Studies on the
functions of the multiple CU pathways present in the genomes of
other bacterial pathogens have also found redundancy and have
FIG 6 Binding of Y. pestis usher deletion mutants to murine bone marrow-
derived macrophages. muBMDM (A) or huMDM (B) were treated with 5 ?g
of cytochalasin D/ml for 1 h and then infected with KIM6? WT, usher dele-
tion mutants, or complemented strains as described in Fig. 4. The results
P ? 0.001 [for comparison of each strain with the WT]).
Hatkoff et al.
iai.asm.org Infection and Immunity
functions of the encoded pili during pathogenesis (64, 70).
In conclusion, we have identified roles for three of the addi-
Our results support a model whereby loci y0348-0352 and y1858-
1862 assemble adhesive pili that bind to a general receptor com-
bled by pathway y1869-1873 bind to a receptor expressed only by
macrophages. The three CU pathways appear to make specific
contributions during pneumonic plague, of which binding to
macrophages conferred by pathway y1869-1873 may be particu-
larly important. Adhesion mediated by the pili could aid in colo-
nization of specific sites within the lung and facilitate the delivery
of Yops into host cells by the Yersinia T3SS. Adhesion to mobile
host cells such as macrophages could also enhance dissemination
of Y. pestis from the lung to secondary sites of infection, promot-
ing the development of systemic disease.
We thank Steven Park, David S. Perlin, and the staff of the University of
Medicine and Dentistry of New Jersey Regional Biocontainment Labora-
tory for performing the Y. pestis mouse infection experiments. We thank
(Stony Brook University) for isolation of muBMDM. We thank Shan Lu
(University of Massachusetts), Susan Straley (University of Kentucky),
of Michigan) for helpful discussions and critical reading of the manu-
This study was supported by Public Health Service grant AI055621
from the National Institute of Allergy and Infectious Diseases (NIAID).
M.H. was supported by grant T32 AI007539 from the NIAID. The Re-
gional Biocontainment Laboratory is supported by Funding from the
Northeast Biodefense Center U54-AI057158-Lipkin and the Northeast
Biodefense Center Animal Core (Perlin).
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